Hepatocyte growth factor--induced proliferation and differentiation of erythroid cells

ABSTRACT

Hepatocyte growth factor stimulates proliferation and differentiation of hematopoietic cells, most preferably burst-forming unit-erythroid cells. The hepatocyte growth factor may be obtained from cells transformed with human gene sequences coding therefor. Pharmaceutical compositions useful to induce the proliferation and differentiation of hematopoietic cells may contain hepatocyte growth factor as an active principle in admixture with a suitable carrier. The pharmaceutical compositions may additionally contain stem cell factor as an active principle.

This application is a 371 of PCT/EP95/04589.

FIELD OF THE INVENTION

The present invention relates to the use of the hepatocyte growth factorfor the preparation of medicaments useful to induce proliferation anddifferentiation of hematopoietic cells, particularly multipotent anderythroid hematopoietic cell progenitors.

BACKGROUND OF THE INVENTION

Hepatocyte Growth Factor (HGF, the same abbreviation having also beenused to define a completely different substance, i.e. hemopoiesis Growthfactor) described by Nakamura et al., 1989, and by Hiyazawa et al.,1989), also known as Scatter Factor (Naldini et al., 1991a; Weidner etal., 1991), has the unique feature of combining mitogenic and motogenicactivities on its target cells. HGF is mitogenic for hepatocytes(Michalopoulos, 1990) and other epithelial cells, such as kidney tubularepithelium, melanocytes and keratinocytes (Kan et al., 1991; Rubin etal., 1991; Halaban et al., 1992; Matsumoto et al., 1991). In thesecells, HGF also promotes "scattering" (Stoker et al., 1987; Gherardi etal., 1989; Weidner et al., 1990, 1991; Naldini et al., 1991a) and matrixinvasion (Weidner et al., 1990; Naldini et al., 1991a), and haschemotactic properties (Morimoto et al., 1991; Giordano et al., 1993).The factor stimulates extracellular matrix (ECM) degradation, byenhancing the synthesis of enzymes involved in ECM proteolysis (Pepperet al., 1992; Boccaccio et al., 1994). HGF acts as a morphogen inneuro-ectodermal development in vivo (Stern et al., 1990), and inducesthree-dimensional organization of epithelial cells in vitro (Montesanoet al., 1991). The factor also promotes the progression of carcinomacells toward malignant invasive phenotypes (Weidner et al., 1990).

The receptor for HGF is the tyrosine kinase encoded by the METproto-oncogene (Naldini et al., 1991a, 1991b; Bottaro et al., 1991), a190 kDa heterodimer of an extracellular 50 kDa α subunit,disulfide-linked to a transmembrane 145 kDa β subunit (Park et al.,1987; Giordano et al., 1989a) Both subunits derive from glycosylationand proteolytic cleavage of a 170 kDa single chain precursor (Giordanoet al., 1989b).

The HGF receptor is expressed in adult epithelial tissues, includingliver, intestine and kidney (Prat et al., 1991a; Di Renzo et al., 1991).It has been reported to be expressed in early stages of development ofepithelial organs (Sonnenberg et al., 1993), and it is oftenoverexpressed in epithelial cancer (Prat et al., 1991a; Di Renzo et al.,1991). We have shown that the receptor is also expressed in endothelialcells and that HGF is a potent angiogenic factor both in vitro and invivo (Bussolino et al., 1992; Grant et al., 1993). The HGF receptor isalso known to be expressed in some populations of blood cells, such asmacrophages, but the meaning of such an expression, which is barelydetectable in the absence of activation, has not been elucidated (Galiniet al., 1993).

EP-A-0,492,614 discloses the use of HGF as a growth enhancer forepitheliocytes, whereas WO 93/08821 discloses the use of HGF for theprevention of the side-effects of chemotherapeuticals.

Now it has been found that the hepatocytes growth factor inducesproliferation and differentiation of multipotent and erythroidhematopoietic progenitors.

Hematopoietic cell growth and differentiation is under the control of acomplex network of cytokines, which act on their target cells viaspecific receptors (Metcalf, 1984; Clark and Kamen, 1987).Erythropoiesis is a complex process in which a specific genetic programis primed (commitment) and executed (maturation). Although much is knownabout maturation, most of the molecular events occurring during thecommitment phase are still obscure. Growth and differentiation oferythroid precursor are regulated by humoral factors and by largelyuncharacterized cell-cell interactions with bone marrow stroma, theso-called hematopoietic microenvironment. Erythropoietin has long beenconsidered the major factor required for erythropoiesis (Koury andBondurant, 1990), other factors being far less specific (IL-3, GM-CSF,TGF-β; Gasson, 1991; Miyajima et al., 1993; Sporn and Roberts, 1992).HGF represents a novel example of a humoral factor specifically activeon erythropoiesis.

Recently, the synergism between HGF, IL-3 and GF, IL-3 and GM-CSF inpromoting the growth of uncharacterized colonies from unfractionatedmurine bone marrow (Kmiecik et al., 1992) has been described. From saidwork, however, no conclusions about the effect of HGF alone could bededuced; moreover, the results obtained using bone marrow cellsuspensions (including lymphocytes and monocytes-macrophages) could beascribed to an indirect effect, mediated by the production ofhematopoietic cytokines by accessory cells.

The synergism between HGF, IL-3 and GM-CSF, in fact, has not beenconfirmed by the authors of the present invention on isolated coloniesof human hematopoietic cells.

SUMMARY OF THE INVENTION

It has been found that HGF stimulates erythroid and multipotentprogenitors in vitro and that the HGF receptor is expressed in asub-population of adult hematopoietic progenitors (CD34⁺). The obtainedresults, which are reported in detail in the examples, suggest that HGFis a paracrine mediator of stromal-hematopoietic cell interactions, bothduring embryogenesis and adult life. HGF may therefore be one of themolecules mediating developmental signals between microenvironment andhematopoietic progenitors.

As a consequence, HGF can be administered, according to the invention,to patients affected with pathologies in which the stimulation ofhemopoiesis is desirable. Examples of such pathologies include primitiveor secondary cytopenias of various origin. Moreover HGF can be used inall of the radio- or chemo-sensitive neoplastic pathologies in which theuse of bone marrow autologous transplant could be suggested, also forthe mobilization in the bloodstream of the hematopoietic precursors(scatter effect). Particularly, HGF can be used as mobilizer of the bonemarrow precursors in the use of peripheral blood as a source of stemcells for bone marrow transplant. Moreover, HGF may be used, alone or incombination with other factors, for the ex vivo expansion of marrowhematopoietic precursors, in any clinical condition requiring it(Akabutu, J. J., Chan, J. R., Prog. Clin. Biol. Res. 389:399-404).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically illustrates numbers of cell colonies obtained in thepresence of specified substances.

FIG. 2 graphically compares numbers of BFU-E and CFU-GEMM derivedcolonies.

FIG. 3 graphically illustrates numbers of cell colonies obtained In thepresence of stem cell factor.

FIG. 4 presents a colony formation assay on CD34+ cells.

DETAILED DESCRIPTION OF THE INVENTION

For the envisaged therapeutical uses, HGF from different sources can beused, for example from animal or human organs or from prokaryotic oreukaryotic cells transformed with genes coding for HGF. By the term HGF,an activated form of HGF is obviously meant, such as described inExample 1 and 6. The use of human recombinant HGF is preferred, but theinvention also applies to all the possible variants of HGF, includingany deletion and/or substitution mutant forms. The hepatocyte growthfactors will be formulated in dosage forms suitable to theadministration of protein substances. The formulations of the inventiontherefore will be administered preferably by the parenteral route andthey can be prepared using conventional techniques and excipients, asdescribed for example in Remington's Pharmaceutical Sciences Handbook,Mack Pub. Co., N.Y., USA.

Anyhow, other administration routes already suggested for protein activeprinciples, such as the nasal, sublingual, rectal and oral routes,cannot be excluded. For the latter, the active principle will suitablybe protected from metabolic degradation making use of known techniques,for example the inclusion in liposome vesicles. The HGF dosage,according to the invention, may vary within wide ranges, for examplefrom about 0.01 mg to about 10 mg of HGF, one or more times daily. Thefollowing examples further illustrate the invention.

EXAMPLE 1

Purification of human recombinant HGF from the Baculovirus expressionsystem.

Full-length HGF cDNA was cloned from human liver mRNA and inserted intothe Baculovirus transfer vector PVL1393 (Invitrogen, San Diego, Calif.).The recombinant vector was cotransfected with the Bsul-digested BacPak6viral DNA (Clontech Laboratories, Palo Alto, Calif.) into Spodopterafrugiperda insect cells (Sf9) by the Lipofectin procedure (Gibco-BRL,Gaithersburg, Md.). Positive clones were identified and purified bydot-blot hybridization and plaque assay. The recombinant virus was usedto infect Sf9 cells with dilutions of 10⁻¹, 10⁻², 10⁻³, 10⁻⁶. After oneweek, the infected cell extracts were blotted on a nylon filter andprobed with radiolabelled full-length human HGF cDNA. The virusescontaining the HGF cDNA gene were subsequently purified by plaque assay.Single viral clones were isolated and used for large scale infection ofSf9 cells.

The recombinant factor was purified by affinity chromatography onheparin (BioRad Laboratories, Hercules, Calif.), according to theprocedure published by Weidner et al., 1990, with some modifications.Sf9 Spodoptera Frugiperda cells were grown at 27° C. in serum-free SF900medium (Gibco Ltd, Scotland).

Exponentially growing cultures were infected by adding the viral stockin serumfree culture medium, and the cells were grown for 3 days. Theculture medium was then collected and was incubated overnight in thepresence of 3% foetal calf serum at 37° C., to ensure full activation ofthe precursor; it was then spun at 300×g for 15 min, to remove cellulardebris, and cleared by centrifugation at 10,000×g for 1 h. Thesupernatant was buffered to pH=7.4 with TRIS, supplemented with amixture of protease inhibitors (1 mM PMSF, 50 μg/ml leupeptin, 10 μg/mlaprotinin, 4 μg/ml pepstatin) and the detergent CHAPS to a finalconcentration of 0.2% w/v, filtered on a 0.45 μm pore Tuffryn membranefilter (Gelman Sciences, Ann Arbor, Mich.) by vacuum suction, cooled to4° C. and applied to a 5 ml heparin-agarose column assembled in an FPLCapparatus in the cold room with a loading rate of 8 ml/h. The column wassequentially washed with 0.15 M NaCl, 50 mM Tris-HCl pH=7.4, 0.2% CHAPSand 0.5 M NaCl, 50 mM Tris-HCl pH=7.4, 0.2% CHAPS until the eluantabsorbance returned to the baseline. Bound materials were eluted with alinear gradient from 0.5 M to 1.8M NaCl over 8 h in 50 mM Tris-HClpH=7.4, CHAPS 0.2%, with a flow rate of 0.2 ml/min, and 2 ml fractionswere collected. The starting material, the column breakthrough andwashings, and the eluted fractions, were scored for the content of HGFby the MDCK scattering assay (Weidner et al., 1990; Naldini et al.,1992). The fractions containing the peak of HGF activity, eluting atapproximately 1M NaCl, were pooled, concentrated with a diafiltrationdevice with 30,000 molecular weight cut off (Amicon Div., GraceIndustrial, Switzerland), checked for biological activity on MDCK cells,and purity by SDS-PAGE and protein stains, to give pure HGF with anaverage yield of the procedure of 150 μg from 700 ml of culturesupernatant.

EXAMPLE 2

Production of human recombinant pro-HGF in insect cells. HGF cDNA wascloned from human liver mRNA (Naldini et al., 1991a) and inserted as aBamHI-EcoRI fragment into the baculovirus transfer vector PVL1393(Invitrogen). The recombinant vector was co-transfected with theBsuI-digested BacPak6 viral DNA (Clontech) into Spodoptera frugiperdainsect cells (Sf9), by the lipofectin procedure.

Positive viral clones isolated by dot-blot hybridization and plaqueassay were used for large scale infection. HGF was obtained from culturesupernatant of Sf9 infected cells 72 hours post-infection, by affinitychromatography on a heparin-Sepharose FPLC column (BioRad), eluted witha linear 0.5-1.8 M NaCl gradient. The unprocessed recombinant factor(pro.sup.• HGF) was detected by Comassie Blue staining as a band of 90kDa in SDSPAGE. Protein concentration was estimated by Comassie Bluestaining and comparison with a standard curve obtained with increasingamounts of bovine serum albumin.

EXAMPLE 3

Stimulation of erythroid and multipotent hematopoietic progenitors.

The effect of HGF on the growth and differentiation of hematopoieticprogenitors was evaluated in colony formation assays. Heparinizedsamples of bone marrow, fetal umbilical cord blood and adult peripheralblood, obtained from volunteers, were diluted with an equal volume ofPhosphate Buffered Saline (PBS), and separated by Ficoll-Hypaque 1077 SD(Pharmacia) density gradient centrifugation at 550 g for 30 minutes.Light-density mononuclear cells (LD-MC) were collected, washed twice inPBS and resuspended in Iscove Modified Dulbecco's Medium (IMDM) (Gibco)supplemented with 5% Fetal Calf Serum (FCS). Mononuclear adherent cellswere then removed by a two steps incubation of 30 minutes each inplastic flasks at 37° C.

Mononuclear non-adherent cells (MNAC) were incubated withneuraminidase-treated sheep erythrocytes for 15 minutes at 37° C.,centrifuged and incubated for 45 minutes at 4° C. T-lymphocyte-depletedMNAC were separated by Ficoll-Hypaque 1077 SD (Pharmacia) densitygradient centrifugation.

T-lymphocyte-depleted MNAC were then incubated for 45 minutes at 4° C.with the following antibodies: anti-CD3, anti-CD4, anti-CD8, antiCD11,anti-CD19, anti-CD57; most of the remaining B- and T- lymphocytes,monocytes and granulocytes were thus removed by incubation for 45 min.at 4° C. with immunomagnetic beads coated with anti-mouse IgG (M-450Dynabeads, Dynal), subsequently collected by a magnet (MPC-1 Dynabeads,Dynal).

A positive selection of the CD34+ cells was then performed: cells wereincubated with an antibody anti-CD34 (My-10; Technogenetics) for 45minutes at 4° C., then for 45 minutes at 4° C. with immunomagnetic beadscoated with anti-mouse IgG; a 4:1 beads/cell ratio was found to providethe best recovery. CD34+ cells bound to the beads were then collected bya magnet and resuspended in IMDM supplemented with 10% FCS. An overnightincubation at 37° C. was then performed; to allow CD34+ cellsdetachment, the beads were subjected to shearing forces by repeatedflushing through a Pasteur pipette. Further details about thenegative/positive double selection procedure used have been publishedpreviously (Bagnara et al., 1991). The recovered cells weremorphologically unidentifiable blast elements on May-Grunwald-Giemsastaining, slightly contaminated by promyelocytes. Flow cytometryanalysis indicated that the percentage of CD34+ cells in the selectedcell preparations varied between a minimum of 30% (when the startingmaterial was bone marrow) and a maximum of 50% (when the startingmaterial was peripheral blood). Contamination by CD4+, CD2+, CD16+ orCD19+ cells was constantly below 1%.

The colony assay for erythroid Burst-Forming Units and for multipotentColony-Forming Units (CFU-GEMM) was performed according to Iscove etal., 1974. Cord blood, bone marrow or peripheral blood CD34+ cells wereplated in 24-well cell culture clusters (Costar), at a density of2.5×10³ cells/well, in a medium containing IMDM, 30% FCS, 2×10⁻⁴ Mhemin, 5×10⁻⁵ β-mercaptoethanol and 0.9% methylcellulose. The cells werestimulated with the following growth factors alone or in combination:Epo 2 ng/ml, IL-3 2 ng/ml, GH-CSF 50 ng/ml, SCF 20 ng/ml, pro-HGF 2, 10or 40 ng/ml. Colonies scored positive only when dark-red and containingmore than four aggregates.

The assay for the 14-day Granulo-Monocyte Colony-Forming Units (CFU-GM)was performed as previously described (Iscove et al., 1971). Cord blood,bone marrow or peripheral blood CD34+ cells were plated in 24-well cellculture clusters (Costar), at a density of 2.5×10³ cells/well, in amedium containing IMDM, 20% FCS, 0.3% Noble agar (Difco) and thefollowing growth factors alone or in combination: IL-3 2 ng/ml, GM-CSF50 ng/ml, SCF 20 ng/ml, pro-HGF 2, 10 or 40 ng/ml.

For the Megakaryocyte Colony-Forming Unit (CFU-meg) assay, plasma clotassay was performed according to Vainchenker et al., 1979. Cord blood,bone marrow or peripheral blood CD34+ cells were plated in 24-well cellculture clusters (Costar), at a density of 2.5×10³ cells/well, in amedium containing IMDM, 20 mg/ml L-Asparagine (Sigma), 3.4 mg/ml CaCl₂,10% bovine plasma citrated (GIBCO), 1% detoxified bovine serum albumin(BSA, fraction V Chon) (Sigma), 10% of heat-inactivated human AB serumand the following growth factors alone or in combination: IL-3 2 ng/ml,GM-CSF 50 ng/ml, SCF 20 ng/ml, pro-HGF 2, 10 or 40 ng/ml. After 12 daysof incubation, the plasma clot was fixed in situ with methanol-acetone1:3. for 20 minutes, washed with PBS and air dried. Fixed plates werestored at -20° C. until immunofluorescence staining was performed;CFU-meg colonies were scored as aggregates of 3-100 cells intensivelyfluorescent to monoclonal antibody CD41 (Immunotech) directed againstthe IIb/IIIa glycoprotein complex. Binding was shown byfluorescein-conjugated goat anti-mouse Ig (Becton Dickinson).

The results are schematized in FIGS. 1 and 2, and they show that, in thepresence of standard concentrations of erythropoietin (2 ng/ml), HGFdramatically increased the number of colonies derived from the BFU-Eprecursors (FIG. 1). HGF also stimulated the growth of colonies derivedfrom multipotent CFU-GEMM progenitors. The number of colonies wascomparable to that obtained by combining known hemopoietic factors suchas GM-CSF and Interleukin-3 (Gasson, 1991; Miyajima et al., 1993). Itshould be noted, however, that the HGF effect was restricted to thestimulation of CFU-GEMM and BFU-E.

Neither granulo-monocytic nor megakaryocytic colonies were ever observedin response to HGF.

The response to HGF was dose-dependent and could be observed atconcentrations of HGF as low as 5 pM both in erythroid and multipotentcolonies (FIG. 2).

The HGF action was also studied on CD34+ foetal hematopoieticprogenitors, enriched from human umbilical cords blood. It is known thatthis population contains a percentage of primitive stem cells higherthan the population purified from adult bone marrow or peripheral blood(Broxmeyer et al., 1992; Lu et al., 1993). As observed in the case ofadult hematopoietic progenitors, HGF stimulated both BFU-E and CFU-GEMMderived colonies.

In the presence of both HGF and Stem Cell Factor (SCF), a significantincrease in the number of CFU-GEMM-derived colonies was observed (FIG.3). In this case, fewer erythroid colonies could be seen compared tothose developed in the cultures stimulated by HGF alone. This suggeststhat the combination of HGF and SCF preferentially affects proliferationof multipotent progenitors.

The erythroid colonies grown in the presence of both growth factors wereextremely large and showed a high hemoglobin content. The size ofCFU-GEMM derived colonies grown in these conditions was also increasedand, within each colony, the erythroid lineage was predominant.

In these assays HGF did not synergize with GM-CSF and Interleukin-3,either tested individually or in combination.

EXAMPLE 4

Expression of the HGF receptor in a subpopulation of adult hematopoieticprogenitors (CD34+).

The presence of HGF receptor at the surface of hematopietic progenitorswas studied by flow cytometry analysis of bone marrow and peripheralblood mononuclear cells. Monoclonal antibodies directed againstextracellular epitopes of the HGF receptor β chain were used. A smallbut clearly identifiable subpopulation of bone marrow cells stainedpositive for the HGF receptor (Table).

    ______________________________________                                                     Phenotype Positive Cells %                                       ______________________________________                                        A.  Unfractionated HGF-R+      0.6 ± 0.1                                      bone marrow HGF-R+/CD34+  0.3 ± 0.05                                        HGF-R+/CD34- 0.3 ± 0.1                                                     HGF-R+/SCF-R+ 0.2 ± 0.1                                                    HGF-R+/SCF-R- 0.4 ± 0.1                                                  B. CFU-GEMM-derived HGF-R+ 15.3 ± 1.5                                       colonies                                                                     C. BFU-E-derived HGF-R+ 9.7 ± 1.2                                           colonies                                                                   ______________________________________                                    

About half of the cells expressing the HGF receptor also co-expressedthe CD34 marker and could thus be identified ashematopoietic-progenitors.

As described above, HGF synergized with the SCF in stimulating thegrowth and differentiation of CFU-GEMM derived colonies. In line withthis observation, a subpopulation of cells co-expressing the HGF and theSCF receptors was identified using a monoclonal antibody againstextracellular epitopes of the SCF receptor.

Similar results were obtained by flow cytometry analysis of CD34+progenitors circulating in the peripheral blood.

Flow cytometry analysis with anti-HGF-receptor antibodies was alsoperformed on cells harvested from the colonies developed in vitro inresponse to HGF. The Table shows that HGF-receptor positive cells werepresent.

EXAMPLE 5

Expression of HGF and its receptor during the embryonal development ofhematopoietic cells.

The expression of the HGF receptor was studied in embryonalhematopoietic cells by in situ hybridization of histological sections ofmouse embryos. Using an antisense MET probe, the HGF receptor mRNA couldbe clearly detected in megaloblastic cells located within the cavity ofthe developing heart and aorta from 10-10.5 days post coitum. SpecificmRNA could be detected in the hepato/biliary primordium, which at thisstage contains hemopoietic precursors. In this developing organerythroid islands showed a higher levels of HGF receptor mRNA, comparedwith the level of expression observed in the surrounding hepatocytes.From 11 days post coitum the hematopoietic embryonal liver alsoexpressed HGF mRNA.

EXAMPLE 6

In order to prove the mobilization of the bone marrow hemopoieticprecursors at the peripheral blood, the murine model has been used.Balb/C mice were treated subcutaneously for 4 days with HGF at variedconcentrations or with control preparations. At the end of thetreatment, mice were killed, the circulating leukocytes were counted andhemopoietic colonies from peripheral blood were cultured. In HGF-treatedmice, contrary to the untreated controls, an about 60% increase incirculating leukocytes was observed as well as an increase in thecolonies obtainable from peripheral blood. This phenomenon has anintensity comparable with that of G-CSF, already described and used tomobilize bone marrow hematopoietic precursors (Janssen, W. E., et al.,Prog. Clin. Biol. Res. 389:429-39).

EXAMPLE 7

Using in the colony formation assay on CD34⁺ cells of Example 3equimolecular amounts of activated HGF, obtained according to Example 1,instead of pro-HGF, statistically similar results have been obtained inthe colonies count as shown in the enclosed FIG. 4.

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I claim:
 1. A method of stimulating proliferation and differentiation ofburst-forming unit-erythroid (BFU-E) cells in vitro which comprisestreating said cells with an effective amount of hepatocyte growth factor(HGF).
 2. The method according to claim 1, wherein the hepatocyte growthfactor is obtained from cells transformed with a human gene sequencecoding for HGF.
 3. The method of claim 1, wherein HGF is used in aconcentration of at least 5 pM.